Protein Complex Of Photosystem Research Articles

Isolation of a novel heterodimeric PSII complex via strep-tagged PsbO

The multi-subunit membrane protein complex photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag. Three distinct PSII complexes were separated by ion-exchange chromatography after the initial affinity purification. Two complexes differ in their oligomeric state (monomeric and dimeric) but share the typical subunit composition of mature PSII. They are characterized by the very high oxygen evolving activity of approx. 6000 μmol O2·(mg Chl·h)−1. Analysis of the third (heterodimeric) PSII complex revealed lower oxygen evolving activity of approx. 3000 μmol O2·(mg Chl·h)−1 and a manganese content of 2.7 (±0.2) per reaction center compared to 3.7 (±0.2) of fully active PSII. Mass spectrometry and time-resolved fluorescence spectroscopy further indicated that PsbO is partially replaced by Psb27 in this PSII fraction, thus implying a role of this complex in PSII repair.

A new platform for development of photosystem I based thin films with superior photocurrent: TCNQ charge transfer salts derived from ZIF-8.

The transmembrane photosynthetic protein complex Photosystem I (PSI) is highly sought after for incorporation into biohybrid photovoltaic devices due to its remarkable photoactive electrochemical properties, chiefly driving charge separation with ∼1 V potential and ∼100% quantum efficiency. In pursuit of these integrated technologies, three factors must be simultaneously tuned, namely, direct redox transfer steps, three-dimensional coordination and stabilization of PSI aggregates, and interfacial connectivity with conductive pathways. Building on our recent successful encapsulation of PSI in the metal–organic framework ZIF-8, herein we use the zinc and imidazole cations from this precursor to form charge transfer complexes with an extremely strong organic electron acceptor, TCNQ. Specifically, the PSI-Zn-H2mim-TCNQ charge transfer salt complex was drop cast on ITO to form dense films. Subsequent voltammetric cycling induced cation exchange and electrochemical annealing of the film was used to enhance electron conductivity giving rise to a photocurrent in the order of 15 μA cm−2. This study paves the way for a myriad of future opportunities for successful integration of this unique class of charge transfer salt complexes with biological catalysts and light harvesters.

3D printed droplet generation devices for serial femtosecond crystallography enabled by surface coating.

The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X-ray free-electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X-ray beam using a liquid jet injector. Owing to the pulsed nature of the X-ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two-photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long-term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal-laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates.

Catalyst: Chemistry’s Role in Providing Clean and Affordable Energy for All

Image Leif Hammarstrom is a professor of chemical physics at Uppsala University, Sweden. He is one of the leaders of the Swedish Consortium for Artificial Photosynthesis, founded in the mid-1990s. He is chair of the Swedish Solar Energy Platform and represents Uppsala University as a core member of the Solar Fuels Institute.

Orientational Control of Electroactive Photosystem I on Chemically Modified Electrodes

The ability to control the orientation of proteins on chemically modified electrodes and within films of conducting or redox polymers would allow for the efficient electron transfer. Unfortunately, extraction of proteins and protein complexes like photosystem I from biological membranes often scrambles the precise orientational control found in native plants. In this talk, we will present alternative methods and chemical reactions that can help maintain the memory of the correct protein orientation as originally in the membrane after the protein complex of photosystem I is extracted from spinach. The resulting voltammetry and photochronoamperometry of these modified electrodes will be compared to non-modified protein extractions.

Serial Femtosecond Crystallography Opens a New Era in Membrane Protein Structural Biology

Serial Femtosecond Crystallography (SFX) provides a novel concept for structure determination, where X‐ray diffraction “snapshots” are collected from a fully hydrated stream of nanocrystals, using femtosecond pulses from the world's first high energy X‐ray free‐electron laser, the Linac Coherent Light Source. The large membrane protein complex Photosystem I was used as the model system for the first serial femtosecond crystallography experiments that showed the proof of concept that diffraction of nanocrystals can be observed using femtosecond pulses that are 1012 stronger than 3rd generation synchrotron sources and destroy any material that is placed in its focus [1],[2]. By using femtosecond pulses briefer than the time‐scale of most damage processes, femtosecond nanocrystallography overcomes the problem of X‐ray damage in crystallography [3] and extends to atomic resolution [4],[5]. It is also applied to membrane proteins crystallized in lipidic environments [6],[7], and has recently led to the structure determination of the Angiotensin receptor which regulates the blood sugar level and is one of the major targets for drugs fighting hypertension [8] and the structure of the rhodopsin‐arrestin complex [9]. Femtosecond crystallography also opens a new avenue for determination of protein dynamics. First experiments on the proof of principle for time resolved serial femtosecond nanocrystallography have been performed on Photosystem I‐ferredoxin [10] and Photosystem II nano‐crystals [11] and conformational changes of the Mn4Ca cluster and its protein environment were observed for the first time in the transition from the dark to the double excited state [12]. Very recently we were able to show that TR‐SFX studies extend to atomic resolution using the photoactive yellow protein as a model system [13]. This pioneering work paves the way for the determination of molecular movies of the dynamics of membrane proteins “at work” in the future including the determination of molecular movies of water splitting.

Crystallography: Resolution beyond the diffraction limit.

A method has been devised that extends the resolution of X-ray crystal structures beyond the diffraction limit. This might help to improve the visualization of structures of proteins that form 'poorly diffracting' crystals. See Letter p.202 Protein structure determination by X-ray crystallography is often limited by lack of access to high-quality crystals that generate sufficiently detailed diffraction patterns. However, X-ray patterns usually also contain continuous diffraction, which is largely ignored but could in principle provide sufficient information to overcome this limitation. Kartik Ayyer and colleagues now show that the continuous diffraction arising from lattice disorder indeed enables structure determination. They use data collected from imperfect crystals of the protein complex photosystem II to obtain an image at 3.5 A resolution. The method puts great value in commonly encountered imperfect crystals, and is expected to enable direct high-resolution structure determination for a range of macromolecular systems.

Macromolecular diffractive imaging using imperfect crystals.

The three-dimensional structures of macromolecules and their complexes are predominantly elucidated by X-ray protein crystallography. A major limitation is access to high-quality crystals, to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields sufficiently high-resolution information that the crystal structure can be solved. The observation that crystals with shrunken unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks1,2 hints that crystallographic resolution for some macromolecules may be limited not by their heterogeneity but rather by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern, equal to the incoherent sum of diffraction from rigid single molecular complexes aligned along several discrete crystallographic orientations and hence with an increased information content3. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins4 —they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 Å limit of measurable Bragg peaks, which allows us to directly phase5 the pattern. With the molecular envelope conventionally determined at 4.5 Å as a constraint, we then obtain a static image of the photosystem II dimer at 3.5 Å resolution. This result shows that continuous diffraction can be used to overcome long-supposed resolution limits of macromolecular crystallography, with a method that puts great value in commonly encountered imperfect crystals and opens up the possibility for model-free phasing6,7.

All-atom molecular dynamics simulation of a photosystem i/detergent complex.

All-atom molecular dynamics (MD) simulation was used to investigate the solution structure and dynamics of the photosynthetic pigment-protein complex photosystem I (PSI) from Thermosynechococcus elongatus embedded in a toroidal belt of n-dodecyl-β-d-maltoside (DDM) detergent. Evaluation of root-mean-square deviations (RMSDs) relative to the known crystal structure show that the protein complex surrounded by DDM molecules is stable during the 200 ns simulation time, and root-mean-square fluctuation (RMSF) analysis indicates that regions of high local mobility correspond to solvent-exposed regions such as turns in the transmembrane α-helices and flexible loops on the stromal and lumenal faces. Comparing the protein-detergent complex to a pure detergent micelle, the detergent surrounding the PSI trimer is found to be less densely packed but with more ordered detergent tails, contrary to what is seen in most lipid bilayer models. We also investigated any functional implications for the observed conformational dynamics and protein-detergent interactions, discovering interesting structural changes in the psaL subunits associated with maintaining the trimeric structure of the protein. Importantly, we find that the docking of soluble electron mediators such as cytochrome c6 and ferredoxin to PSI is not significantly impacted by the solubilization of PSI in detergent.

Microcrystallization techniques for serial femtosecond crystallography using photosystem II from Thermosynechococcus elongatus as a model system.

Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano- or microcrystals of biomolecules in their mother liquor using high-energy, X-ray free-electron lasers. The success of SFX experiments strongly depends on the ability to grow large amounts of well-ordered nano/microcrystals of homogeneous size distribution. While methods to grow large single crystals have been extensively explored in the past, method developments to grow nano/microcrystals in sufficient amounts for SFX experiments are still in their infancy. Here, we describe and compare three methods (batch, free interface diffusion (FID) and FID centrifugation) for growth of nano/microcrystals for time-resolved SFX experiments using the large membrane protein complex photosystem II as a model system.

Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel

Traditional macroscale protein crystallization is accomplished nontrivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time-consuming and resource-intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these "difficult to crystallize" proteins such as femtosecond nanocrystallography are being developed yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via second-order nonlinear imaging of chiral crystals (SONICC) was applied for the detection of nanometer- and micrometer-sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.

Dielectrophoretic Sorting of Membrane Protein Nanocrystals

Structure elucidation of large membrane protein complexes is still a considerable challenge, yet is a key factor in drug development and disease combat. Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals, thus overcoming the experimental riddle faced in traditional crystallography methods. Here, we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis. We demonstrate the excellent sorting power of this new approach with numerical simulations of selected submicrometer beads in excellent agreement with experimental observations. Crystals from batch crystallization broths of the huge membrane protein complex photosystem I were sorted without further treatment, resulting in a high degree of monodispersity and crystallinity in the ~100 nm size range. Microfluidic integration, continuous sorting, and nanometer-sized crystal fractions make this method ideal for direct coupling to femtosecond nanocrystallography.

Structural Changes of the Oxygen-evolving Complex in Photosystem II during the Catalytic Cycle

The oxygen-evolving complex (OEC) in the membrane-bound protein complex photosystem II (PSII) catalyzes the water oxidation reaction that takes place in oxygenic photosynthetic organisms. We investigated the structural changes of the Mn4CaO5 cluster in the OEC during the S state transitions using x-ray absorption spectroscopy (XAS). Overall structural changes of the Mn4CaO5 cluster, based on the manganese ligand and Mn-Mn distances obtained from this study, were incorporated into the geometry of the Mn4CaO5 cluster in the OEC obtained from a polarized XAS model and the 1.9-Å high resolution crystal structure. Additionally, we compared the S1 state XAS of the dimeric and monomeric form of PSII from Thermosynechococcus elongatus and spinach PSII. Although the basic structures of the OEC are the same for T. elongatus PSII and spinach PSII, minor electronic structural differences that affect the manganese K-edge XAS between T. elongatus PSII and spinach PSII are found and may originate from differences in the second sphere ligand atom geometry.

Two-dimensional electronic spectroscopy for mapping molecular photophysics

We demonstrate the ability of two-dimensional electronic spectroscopy (2DES) to map ultrafast energy transfer and dynamics in two systems: the pigment–protein complex photosystem I (PSI) and aggregates of the conjugated polymer poly(3-hexylthiophene) (P3HT). A detailed description of our experimental set-up and data processing procedure is also given.

Photoreduction of Catalytic Platinum Particles Using Immobilized Multilayers of Photosystem I

Using the abundance of available electrons generated by immobilized multilayers of the photoactive protein complex Photosystem I (PSI), we have photoreduced platinum particles that are catalytically active for the H(2)/H(+) redox couple. The resulting platinized PSI films were optimized using electrochemical measurements and then characterized using X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and scanning electrochemical microscopy (SECM). These results demonstrate a novel method for generating immobilized platinum catalysts that are readily available on the surface of a photoactive PSI multilayer.

Role of Novel Dimeric Photosystem II (PSII)-Psb27 Protein Complex in PSII Repair

The multisubunit membrane protein complex Photosystem II (PSII) catalyzes one of the key reactions in photosynthesis: the light-driven oxidation of water. Here, we focus on the role of the Psb27 assembly factor, which is involved in biogenesis and repair after light-induced damage of the complex. We show that Psb27 is essential for the survival of cyanobacterial cells grown under stress conditions. The combination of cold stress (30 °C) and high light stress (1000 μmol of photons × m(-2) × s(-1)) led to complete inhibition of growth in a Δpsb27 mutant strain of the thermophilic cyanobacterium Thermosynechococcus elongatus, whereas wild-type cells continued to grow. Moreover, Psb27-containing PSII complexes became the predominant PSII species in preparations from wild-type cells grown under cold stress. Two different PSII-Psb27 complexes were isolated and characterized in this study. The first complex represents the known monomeric PSII-Psb27 species, which is involved in the assembly of PSII. Additionally, a novel dimeric PSII-Psb27 complex could be allocated in the repair cycle, i.e. in processes after inactivation of PSII, by (15)N pulse-label experiments followed by mass spectrometry analysis. Comparison with the corresponding PSII species from Δpsb27 mutant cells showed that Psb27 prevented the release of manganese from the previously inactivated complex. These results indicate a more complex role of the Psb27 protein within the life cycle of PSII, especially under stress conditions.

Femtosecond X-ray protein nanocrystallography

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

Redox control of oxoiron(IV)

The oxygen-evolving centre in the protein complex photosystem II involves Ca2+ within a high-valent oxomanganese cluster, but the role of Ca2+ has yet to be clarified. Interestingly, the electron-transfer behaviour of a non-haem oxoiron(IV) complex has now been found to be significantly altered by Lewis acid metal-ion binding to the oxo atom.

Functionalized Nanoporous Gold Leaf Electrode Films for the Immobilization of Photosystem I

Plants and some types of bacteria demonstrate an elegant means to capitalize on the superabundance of solar energy that reaches our planet with their energy conversion process called photosynthesis. Seeking to harness Nature's optimization of this process, we have devised a biomimetic photonic energy conversion system that makes use of the photoactive protein complex Photosystem I, immobilized on the surface of nanoporous gold leaf (NPGL) electrodes, to drive a photoinduced electric current through an electrochemical cell. The intent of this study is to further the understanding of how the useful functionality of these naturally mass-produced, biological light-harvesting complexes can be integrated with nonbiological materials. Here, we show that the protein complexes retain their photonic energy conversion functionality after attachment to the nanoporous electrode surface and, further, that the additional PSI/electrode interfacial area provided by the NPGL allows for an increase in PSI-mediated electron transfer with respect to an analogous 2D system if the pores are sufficiently enlarged by dealloying. This increase of interfacial area is pertinent for other applications involving electron transfer between phases; thus, we also report on the widely accessible and scalable method by which the NPGL electrode films used in this study are fabricated and attached to glass and Au/Si supports and demonstrate their adaptability by modification with various self-assembled monolayers. Finally, we demonstrate that the magnitude of the PSI-catalyzed photocurrents provided by the NPGL electrode films is dependent upon the intensity of the light used to irradiate the electrodes.

The mechanism of photosynthetic water splitting

Oxygenic photosynthesis, which provides the biosphere with most of its chemical energy, uses water as its source of electrons. Water is photochemically oxidized by the protein complex photosystem II (PSII), which is found, along with other proteins of the photosynthetic light reactions, in the thylakoid membranes of cyanobacteria and of green plant chloroplasts. Water splitting is catalyzed by the oxygen-evolving complex (OEC) of PSII, producing dioxygen gas, protons and electrons. O(2) is released into the atmosphere, sustaining all aerobic life on earth; product protons are released into the thylakoid lumen, augmenting a proton concentration gradient across the membrane; and photo-energized electrons pass to the rest of the electron-transfer pathway. The OEC contains four manganese ions, one calcium ion and (almost certainly) a chloride ion, but its precise structure and catalytic mechanism remain unclear. In this paper, we develop a chemically complete structure of the OEC and its environment by using molecular mechanics calculations to extend and slightly adjust the recently-obtained X-ray crystallographic model with reference to this structure and to some important recent experimental results.